ENZYMES

-Keshava Pavan K

• Prosthetic group

– tightly bound coenzymes

– FAD, FMN, Biotin

• Cosubstrates

– loosely bound coenzymes

– NAD+, NADP+

• Coenzymes transferring H :

– FAD, FMN, NAD, NADP- water soluble vitamins

– Tetrahydrobiopterin

– Lipoic acid

– Coenzyme Q

• Coenzymes transferring groups other than H

– Biotin (CO2)

– Pyridoxal phosphate (amino)

– CoA (acyl)

– Tetrahydro folic acid (1 C groups)

– Methyl cobalamine (CH3)

– ATP (PO4)

– S Adenosyl methionine (CH3)

– UDP (glucose/galactose)

• Last three do not belong to Vitamin B complex

• Activators are metal ions

– tightly bound- metalloenzymes

– loosely bound- metal activated enzymes

• Stabilise proper conformation

• Cu2+ for tyrosinase, Mg2+ for kinases

• Fe, Cu in oxidation-reduction reactions

• Pyruvate dehydrogenase complex requires 5 activators: Mg2+, NAD+, FAD+, TPP, CoA

• Xanthine oxidase requires FAD, Mo, Fe

• Multifunctional enzymes: same enzyme molecule having different functions for different parts of that enzyme molecule

– fatty acid synthase complex

• Multienzyme complex: many enzymes clustered together having common function

• Active site of lysozyme:

Glu Asp Trp Trp Asp

35 52 62 63 101

Catalytic site substrate binding site

• Hexokinase- 0.02 mmol L-1 : always active

• Glucokinase- 10 mmol L-1 : only when glucose concentration is high, only in liver

CLINICAL APPLICATIONS OF COMPETITIVE INHIBITION

• Sulphonamides(NH2-C6H5-SO2-NH2) resemble PABA (NH2-C6H5-COOH), hence inhibits it

• Anticancer drugs like methotrexate, aminopterin inhibit dihydrofolate reductase

• Alcohol dehydrogenase catalyses conversion of methyl alcohol to formaldehyde. Ethyl alcohol inhibits it. Therefore ethyl alcohol is used during methyl alcohol poisoning.

• Allopurinol is used to treat gout as it inhibits xanthine oxidase that converts hypoxanthine to uric acid

• Dicoumarol is an anticoagulantas it inhibits Vit K needed for coagulation

• Lovastatin inhibits HMG-CoA reductase, hence used to reduce cholesterol level in blood

NONCOMPETITIVE INHIBITORS

• Cyanide inhibits cytochrome oxidase

• Iodoacetate inhibits enzynes having –SH (sulfhydryl) group at active site like glyceraldehyde-3-phosphate dehydrogenase

• Fluoride inhibits enolase (binding with Mg 2+

or Mn2+)

• Di-isopropyl fluorophosphate (DFP) inhibits enzymes having serine at active site like chymotrypsin, acetylcholine esterase

SUICIDE INHIBITION

• Irreversible

• Mechanism based inhibition

• Allopurinol becomes alloxanthine which is a more potent inhibitor

• 5-fluoro uracil becomes fluoro deoxy uridylate which binds to the enzyme thymidylate synthetase and inhibits it

ALLOSTERIC MODULATION

• Regulation of enzyme activity in the body

• Positive & negative modulators

• Not a substrate analog

• Reversible

• Allosteric site other than active site

• Brings about conformational change

• Mostly oligomeric enzymes – Aspartate transcarbamoylase (6 polypeptide chains)

– Pyruvate kinase (4 polypeptide chains)

• Have sigmoidal curve

ENZYME INHIBITOR ACTIVATOR

ALA synthase heme

phosphofructokinase ATP, citrate AMP

Aspartate transcarbamoylase CTP ATP

UNCOMPETITIVE INHIBITION

• Cannot bind with free enzyme, only to E-S complex

• 1/V

1/S

• Eg,. Inhibition of placental alkaline phosphatase (Regan isoenzyme) by phenyl alanine

inhibitor

REGULATION OF ENZYME ACTIVITY

• Induction (depression) and repression – Some are constitutive enzymes like hexokinase

– Others are inducible like glucokinase

• Allosteric modulation

• Covalent modulation – Zymogen activation, phosphorylation and

dephosphorylation

• Compartmentalisation of pathways

• Degradation

• isoenzymes

DIAGNOSTIC ENZYMES

• Aspartate transaminase

– Normal 2 – 40 IU/L

– Increases during MI & liver diseases

• Alanine transaminase

– Normal 0 – 45 IU/L

– Very high during liver diseases

– Moderately high during MI

• Alkaline phosphatase

– Hydrolysis of phosphate esters

– Optimum pH 9.5-10

– 25-100 IU/L

– Increases in liver & bone diseases

– Very high in cholestatic/obstructive jaundice

• Acid phosphatase (prostatic fraction)

– Optimum pH 4.5

– Increases in bone diseases & prostate cancers

– Prostatic specific antigen-protease

• 5mg/L

• 4 to 10mg/L benign; >10 mg/L cancerous

• 5’ nucleotidase

– Normal 2-10 IU/L

– High in liver diseases, very high in cholestasis

• γ- glutamyl transpeptidase

– Normal 10-30 IU/L

– High in liver diseases, very high in alcoholics

• Cholinesterase

– Normal 2-121 IU/L

– Inhibited by organophosphorous pesticides

• Pseudocholinesterase

– Decreases in liver diseases

• Amylase

– Normal 50-120 IU/L

– Very high in acute pancreatitis

– Moderately high in chronic pancreatitis & mumps

• Lipase

– Normal 0.2-1.5 IU/L

– Diagnostics same as above; also high in cholestasis

THERAPEUTIC ENZYMES

• Streptokinase

– To dissolve intravascular clots

• Asparginase

– To treat leukemia

• Pancreatin

– Lipase, trypsin

– To treat pancreatic insufficiency

• Collagenase

– To remove scar tissue

ANALYTICAL ENZYMES

• Glucose oxidase, peroxidase

– Estimation of glucose

• Cholesterol oxidase

– Estimation of cholesterol

• Horse- raddish peroxidase- ELISA

• Taq polymerase- PCR

• Restriction endonuclease- rDNA technology & DNA fingerprinting

RIBOZYMES

• snRNA/small nuclear RNA

– Splicing

• Peptidyl transferase

– translation